Method for preventing or treating liver tissue damage and associated diseases

ABSTRACT

The present invention relates to the use of plasminogen in the treatment and/or elimination of hepatic injury, thereby providing a new therapeutic strategy for treating different types of hepatic injuries.

TECHNICAL FIELD

The present invention relates to the use of plasminogen or plasmin inthe prevention and/or treatment of hepatic tissue injury caused byvarious reasons, thereby providing a brand new therapeutic strategy fortreating hepatic tissue injury and its related disorders.

BACKGROUND ART

Hepatic injury or hepatic tissue injury is a hepatic parenchymal diseasecaused by various reasons, and is a general term for a series ofpathological changes, such as hepatic tissue inflammation, hepatocytedegeneration, necrosis and hepatic tissue fibrosis. Common causes areinflammation, liver congestion, viral infections, poisoning, drugs,radiation, etc. Some diseases, such as diabetes mellitus, hepatitis,hypertension, and atherosclerosis, also occur with injuries of hepatictissue cells.

Drugs are very common causes of hepatic tissue damage. Common drugs thatcause hepatic tissue damage include: anti-tuberculosis drugs, such asrifampicin, isoniazid, and ethambutol; anti-tumor drugs, such ascyclophosphamide, methotrexate, 5-fluorouracil, carboplatin, andcisplatin; lipid-regulating and lipid-lowering drugs, such as statins(atorvastatin and lovastatin), fenofibrate, clofibrate, and niacin;steroid hormones, such as estrogenic drugs, oral contraceptives, andmale anabolic hormones; cardiovascular drugs, such as amiodarone,warfarin, and calcium ion antagonists; anti-rheumatic drugs, such asantifan, fenbufen, aspirin, and indomethacin; antibiotics, such aschloramphenicol, roxithromycin, ketoconazole, penicillins, andsulfonamides; anti-allergic drugs, such as promethazine (Phenergan),chlorpheniramine (Chlorphenamine), and loratadine (Clarityne);anti-ulcer drugs, such as cimetidine, ranitidine, and famotidine;antifungal drugs, such as ribavirin; and so on.

Alcohol is a great threat to the liver. Long-term or intermittent heavydrinking can cause hepatic tissue injury. The greater the drinkingamount of alcohol is and the longer the drinking history is, the moresevere the consequences are. Alcohol directly poisons hepatocytes andaffects their structures and functions.

Alcoholic hepatic injury is a chronic, toxic hepatic injury, a hepaticdisease caused by long-term heavy drinking. In the early stage, it isusually manifested as fatty liver, which can then develop into alcoholichepatitis, hepatic fibrosis and hepatic cirrhosis. Its main clinicalfeatures include nausea, vomiting, jaundice, and sometimes hepatomegalyand tenderness. Severe alcoholism can induce extensive hepatocytenecrosis or even hepatic failure. Alcoholic hepatopathy is one of commonhepatic diseases in China, and seriously endangers people'shealth^([1]).

In addition to the toxic hepatic injury caused by alcohol, other“hepatotropic poisons” such as chemical toxicants and certain drugs inthe environment can also cause hepatic injury. As an importantdetoxification organ for the human body, the liver has dual blood supplyfrom the hepatic arteries and hepatic veins. Chemical substances canenter the liver through the portal veins of the gastrointestinal tractor the systemic circulation for conversion, and thus the liver isvulnerable to toxic substances in chemicals. There are substances thatare toxic to the liver in both nature and human industrial productionprocesses, known as “hepatotropic poisons”. People are generallysusceptible to these poisons, and they have a short incubation period.The pathological process is directly related to the amount of chemicalsubstances, and these substances may cause different levels ofhepatocyte necrosis, steatosis and hepatic cirrhosis in the liver. Thepathological manifestations include (1) steatosis. Carbon tetrachloride,yellow phosphorus and the like can interfere with the synthesis andtranslocation of lipoproteins, forming fatty liver. (2) Lipidperoxidation, which is a special manifestation form of toxic hepaticinjury. For example, carbon tetrachloride is metabolized in vivo toproduce an intermediate product with strong oxidizing ability, leadingto lipid peroxidation on biofilms, destructing membrane phospholipidsand changing the structures and functions of cells. (3) Cholestasis,which is mainly associated with damage of the hepatic cell membrane andmicrovilli, causing bile acid excretion disorder^([2]).

Radiation can also cause hepatic tissue injury. In general, radiationsources are high-energy electromagnetic waves or high-energy particlesproduced by natural or artificial energy sources. Either instantirradiation with high-dose rays or prolonged irradiation with low-doserays may cause tissue injury. The radiation energy destroys thechromosomes and enzymes of cells and disrupts the normal functions ofcells.

Diabetic hepatic tissue injury refers to the lesions of liver histologyand functions caused by diabetes mellitus. Hepatic injuries known to becaused by diabetes mellitus include: hepatic enzymology abnormalities,which can cause carbon dioxide accumulation, acidosis, reduced oxygensupply and increased oxygen consumption in hepatocytes, resulting inincreased activity of liver transaminases and bilirubin metabolismdisorder, with severe cases causing hepatocyte necrosis; fatty liver,wherein diabetes mellitus is the third most common cause of fatty liveramong all causes of fatty liver, and 21%-78% of diabetics have fattyliver; and hepatitis, cirrhosis and hepatic carcinoma, wherein theprevalence of viral hepatitis in diabetics is about 2-4 times that innormal people, and the incidence of primary hepatic carcinoma is about 4times that in normal people. Diabetic hepatopathy not only damages thequality of life of millions of patients, but also creates care neededfor a huge burden cost and healthcare system strength.

Viral infection of the liver is also a common cause of hepatic injury,such as viral hepatitis B, viral hepatitis C, and viral hepatitis E.

Intrahepatic blood congestion may also cause hepatic tissue injury.Intrahepatic blood congestion is mainly caused by the following factors:hepatic veno-occlusive disease, Budd-Chiari syndrome, chronic rightcardiac insufficiency and constrictive pericarditis.

Any disease that blocks the return of inferior vena cava blood to theheart can lead to liver congestion, such as rheumatic valvular heartdisease, chronic constrictive pericarditis, hypertensive heart disease,ischemic heart disease, pulmonary heart disease and congenital heartdisease.

Congestive hepatic injury initially affects the central lobule region;there are venous congestion and expansion in the central lobules, andthe degree of hepatic sinusoid expansion varies depending on thedistance between the hepatic sinusoids and veins in the central lobules;hepatocytes in the central lobules are compressed, deformed andatrophied; there are granular deformation in the cytoplasm, with nuclearcondensation, nuclear division and cell necrosis, accompanied by brownpigmentation with the brown pigment located in the central lobulespotentially caused by cholestasis; and degeneration and necrosis of thehepatic parenchyma near the central veins is the most serious, necrotictissues extend toward the portal area with the exacerbation ofcongestion, patients with severe congestion only have normal livertissues in the portal area, the reticular fibers around the centralveins can collapse over time, and it can be seen that reticular fibroustissues and fine fiber bundles extend from one central vein to another.

Currently, the treatment of hepatic tissue injury mainly includes thecontrol and treatment of causes, as well as supportive treatment.Scientists have been looking for drugs that have a direct, good repaireffect on injured liver tissues for a long time. The present inventorshave also conducted intensive research on this. It is found throughexperiments that plasminogen, a protein substance naturally present inthe human body, has a good repair effect on hepatic tissue injuriescaused by poisoning, radiation, chemotherapeutic drugs and diabetesmellitus. Plasminogen is expected to become a new strategy for treatinghepatic tissue injury and its related disorders.

Plasminogen (plg) is an inactive precursor of plasmin, a single-strandedglycoprotein that is composed of 791 amino acids and has a molecularweight of about 92 kDa^([3,4]). Plasminogen is mainly synthesized in theliver and is abundantly present in the extracellular fluid. The contentof plasminogen in plasma is about 2 μM. Therefore, plasminogen is a hugepotential source of proteolytic activity in tissues and bodyfluids^([5,6]). Plasminogen exists in two molecular forms: glutamicacid-plasminogen (Glu-plasminogen) and lysine-plasminogen(Lys-plasminogen). The naturally secreted and uncleaved forms ofplasminogen have an amino-terminal (N-terminal) glutamic acid and aretherefore referred to as glutamic acid-plasminogen. However, in thepresence of plasmin, glutamic acid-plasminogen is hydrolyzed tolysine-plasminogen at Lys76-Lys77. Compared with glutamicacid-plasminogen, lysine-plasminogen has a higher affinity for fibrinand can be activated by PAs at a higher rate. The Arg560-Va1561 peptidebond between these two forms of plasminogen can be cleaved by uPA ortPA, resulting in the formation of plasmin as a disulfide-linkeddouble-strand protease^([7]). The amino-terminal portion of plasminogencontains five homotrimeric rings, i.e., the so-called kringles, and thecarboxy-terminal portion contains a protease domain. Some kringlescontain lysine-binding sites that mediate the specific interaction ofplasminogen with fibrin and its inhibitor α2-AP. A newly discovered 38kDa fragment of plasminogen, comprising kringles 1-4, is a potentinhibitor of angiogenesis. This fragment is named as angiostatin and canbe produced by the proteolysis of plasminogen via several proteases.

BRIEF DESCRIPTION OF THE INVENTION

In one aspect, the present invention relates to the use of plasminogenor plasmin in the manufacture of a medicament, article or kit forpreventing and/or treating hepatic tissue injury and its relateddisorders in a subject. The present invention also relates to a methodfor manufacturing a medicament, comprising preparing a medicament,article or kit for preventing and/or treating hepatic tissue injury andits related disorders in a subject using plasminogen together with apharmaceutically acceptable carrier.

In one embodiment, the hepatic tissue injury and its related disordersare hepatic injury and its related disorders caused by radiation orchemical substances. In one embodiment, the radiation or chemicalsubstances causing hepatic injury and its related disorders areradiotherapy or chemotherapy methods and drugs used for the treatment ofcancer. In one embodiment, the radiation is radiation caused by anaccident or other event such as a work environment. In one embodiment,the hepatic tissue injury and its related disorders are toxic hepaticinjury and its related disorders. In one embodiment, the toxic hepaticinjury is toxic hepatic injury caused by “hepatotropic poisons”including alcohol. In one embodiment, the hepatic tissue injury and itsrelated disorders are caused by diabetes mellitus and one of thecomplications of diabetes mellitus. In one embodiment, the hepatictissue injury and its related disorders are due to hepatitis caused byviral infection of the liver, such as hepatitis caused by hepatitis Avirus, hepatitis B virus, hepatitis C virus, hepatitis D virus orhepatitis E virus. In one embodiment, the hepatic tissue injury and itsrelated disorders are drug-induced hepatic injury and its relateddisorders. In one embodiment, the hepatic tissue injury and its relateddisorders are caused by intrahepatic blood congestion (livercongestion). In one embodiment, the hepatic tissue injury and itsrelated disorders are diabetic hepatic injury and its related disorders,toxic hepatic injury and its related disorders, drug-induced hepaticinjury or its related disorders, radiation-induced hepatic injury andits related disorders, viral infectious hepatic injury and its relateddisorders, or congestive hepatic injury and its related disorders. Inone embodiment, the hepatic tissue injury and its related disordersinclude hepatic dysfunction, abnormal hepatic enzymology, liverdiscomfort and haphalgesia, hepatomegaly, splenomegaly,hepatosplenomegaly, hepatitis, fatty liver, cholangitis, hepaticcirrhosis, hepatic necrosis and hepatic carcinoma caused by hepatictissue injury.

In one embodiment, the sequence in this patent application refers to thepatent document CN 102154253 A. In one embodiment, the plasminogen is aprotein that comprises a plasminogen active fragment and still hasplasminogen activity.

In one embodiment, the plasminogen is selected from Glu-plasminogen,Lys-plasminogen, mini-plasminogen, micro-plasminogen, δ-plasminogen orany combination thereof. In one embodiment, the plasminogen or plasminis administered systemically or locally, including topical, intravenous,intramuscular, subcutaneous, inhalation, intraspinal, local injection,intraarticular injection or rectal administration. In one embodiment,the related disorders of diabetic hepatic injury or the relateddisorders of toxic hepatic injury include: abnormal hepatic enzymology,liver discomfort and haphalgesia, hepatomegaly, splenomegaly,hepatosplenomegaly, hepatitis, fatty liver, cholangitis, hepaticcirrhosis, hepatic necrosis and hepatic carcinoma. In one embodiment,the hepatic injury and its related disorders are caused by diabetesmellitus-induced angiopathy of large vessels, small vessels, andmicrovessels. In one embodiment, the plasminogen can be administered incombination with one or more other drugs. In one embodiment, the otherdrugs include: liver-protecting drugs, antidiabetic drugs,antithrombotic drugs, anticoagulant drugs, hypolipidemic drugs, drugsagainst cardiovascular and cerebrovascular diseases, and anti-infectivedrugs.

In one embodiment, the subject is a mammal, preferably human.

In one embodiment, the hepatic injury caused by diabetes mellitus iscaused by diabetes mellitus-induced angiopathy of large vessels, smallvessels, and microvessels.

In one embodiment, the subject has a low level of plasmin orplasminogen. Specifically, the low level is innate, secondary and/orlocal.

In one embodiment, the sequence in this patent application refers to thepatent document CN 102154253 A. In one embodiment, the plasminogen is aprotein that has 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35,1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2 or 1 amino acid added,deleted and/or substituted in SEQ ID No. 2, 6, 8, 10 or 12, and stillhas the activity of plasminogen. In one embodiment, the plasminogen is aprotein that comprises a plasminogen active fragment and still has theactivity of plasminogen. In one embodiment, the plasminogen is selectedfrom Glu-plasminogen, Lys-plasminogen, mini-plasminogen,micro-plasminogen, δ-plasminogen or any combination thereof. In oneembodiment, the plasminogen is a conservatively substituted variantselected from variants of Glu-plasminogen, Lys-plasminogen,mini-plasminogen, δ-plasminogen or micro-plasminogen. In one embodiment,the plasminogen is a human natural plasminogen, such as an ortholog ofplasminogen shown in SEQ ID No. 2, e.g., an ortholog of plasminogen fromprimates or rodents, for example, an ortholog of plasminogen fromgorillas, rhesus monkeys, murine, cows, horses and dogs. Mostpreferably, the amino acid sequence of the plasminogen of the presentinvention is as shown in SEQ ID No. 2, 6, 8, 10 or 12.

In one embodiment, the plasminogen is administered in combination with asuitable polypeptide carrier or stabilizer. In one embodiment, theplasminogen is administered at a dosage of 0.0001-2000 mg/kg, 0.001-800mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg/kg or 10-100mg/kg (by per kg of body weight) or 0.0001-2000 mg/cm², 0.001-800mg/cm², 0.01-600 mg/cm², 0.1-400 mg/cm², 1-200 mg/cm², 1-100 mg/cm² or10-100 mg/cm² (by per square centimeter of body surface area) daily,preferably the dosage is repeated at least once, preferably the dosageis administered at least daily. In the case of local administration, theabove dosages may also be further adjusted depending on thecircumstances.

In one embodiment, the plasminogen is administered by systemic ortopical route, preferably by the following routes: topical, intravenous,intramuscular, subcutaneous, inhalation, intraspinal, local injection,intraarticular injection or rectal route. In one embodiment, the localadministration is performed by applying a plasminogen-containingcatheter in the liver area.

In one aspect, the present invention relates to a method for preventingand/or treating hepatic tissue injury and its related disorders in asubject, comprising administering an effective amount of plasminogen orplasmin to the subject. The present invention also relates to the use ofplasminogen or plasmin for preventing and/or treating hepatic tissueinjury and its related disorders in a subject.

In one embodiment, the hepatic tissue injury and its related disordersare hepatic injury and its related disorders caused by radiation orchemical substances. In one embodiment, the radiation or chemicalsubstances causing hepatic injury and its related disorders areradiotherapy or chemotherapy methods and drugs used for the treatment ofcancer. In one embodiment, the radiation is radiation caused by anaccident event. In one embodiment, the hepatic tissue injury and itsrelated disorders are toxic hepatic injury and its related disorders. Inone embodiment, the toxic hepatic injury is toxic hepatic injury causedby “hepatotropic poisons” including alcohol. In one embodiment, thehepatic tissue injury and its related disorders are caused by diabetesmellitus and one of the complications of diabetes mellitus. In oneembodiment, the hepatic tissue injury and its related disorders are dueto hepatitis caused by viral infection of the liver, such as hepatitiscaused by hepatitis A virus, hepatitis B virus, hepatitis C virus,hepatitis D virus or hepatitis E virus. In one embodiment, the hepatictissue injury and its related disorders are drug-induced hepatic injuryand its related disorders. In one embodiment, the hepatic tissue injuryand its related disorders are caused by intrahepatic blood congestion(liver congestion). In one embodiment, the hepatic tissue injury and itsrelated disorders are diabetic hepatic injury and its related disorders,toxic hepatic injury and its related disorders, drug-induced hepaticinjury or its related disorders, radiation-induced hepatic injury andits related disorders, viral infectious hepatic injury and its relateddisorders, or congestive hepatic injury and its related disorders. Inone embodiment, the hepatic tissue injury and its related disordersinclude hepatic dysfunction, abnormal hepatic enzymology, liverdiscomfort and haphalgesia, hepatomegaly, splenomegaly,hepatosplenomegaly, hepatitis, fatty liver, cholangitis, hepaticcirrhosis, hepatic necrosis and hepatic carcinoma caused by hepatictissue injury.

In one embodiment, the sequence in this patent application refers to thepatent document CN 102154253 A. In one embodiment, the plasminogen is aprotein that comprises a plasminogen active fragment and still hasplasminogen activity. In one embodiment, the plasminogen is selectedfrom Glu-plasminogen, Lys-plasminogen, mini-plasminogen,micro-plasminogen, δ-plasminogen or any combination thereof. In oneembodiment, the plasminogen or plasmin is administered systemically orlocally, including topical, intravenous, intramuscular, subcutaneous,inhalation, intraspinal, local injection, intraarticular injection orrectal administration. In one embodiment, the related disorders ofdiabetic hepatic injury or the related disorders of toxic hepatic injuryinclude: abnormal hepatic enzymology, liver discomfort and haphalgesia,hepatomegaly, splenomegaly, hepatosplenomegaly, hepatitis, fatty liver,cholangitis, hepatic cirrhosis, hepatic necrosis and hepatic carcinoma.In one embodiment, the hepatic injury and its related disorders arecaused by diabetes mellitus-induced angiopathy of large vessels, smallvessels, and microvessels. In one embodiment, the plasminogen can beadministered in combination with one or more other drugs. In oneembodiment, the other drugs include: liver-protecting drugs,antidiabetic drugs, antithrombotic drugs, anticoagulant drugs,hypolipidemic drugs, drugs against cardiovascular and cerebrovasculardiseases, and anti-infective drugs.

In one embodiment, the subject is a mammal, preferably human.

In one embodiment, the hepatic injury caused by diabetes mellitus iscaused by diabetes mellitus-induced angiopathy of large vessels, smallvessels, and microvessels.

In one embodiment, the subject has a low level of plasmin orplasminogen. Specifically, the low level is innate, secondary and/orlocal.

In one embodiment, the sequence in this patent application refers to thepatent document CN 102154253 A. In one embodiment, the plasminogen is aprotein that has 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35,1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2 or 1 amino acid added,deleted and/or substituted in SEQ ID No. 2, 6, 8, 10 or 12, and stillhas the activity of plasminogen. In one embodiment, the plasminogen is aprotein that comprises a plasminogen active fragment and still has theactivity of plasminogen. In one embodiment, the plasminogen is selectedfrom Glu-plasminogen, Lys-plasminogen, mini-plasminogen,micro-plasminogen, δ-plasminogen or any combination thereof. In oneembodiment, the plasminogen is a conservatively substituted variantselected from variants of Glu-plasminogen, Lys-plasminogen,mini-plasminogen, δ-plasminogen or micro-plasminogen. In one embodiment,the plasminogen is a human natural plasminogen, such as an ortholog ofplasminogen shown in SEQ ID No. 2, e.g., an ortholog of plasminogen fromprimates or rodents, for example, an ortholog of plasminogen fromgorillas, rhesus monkeys, murine, cows, horses and dogs. Mostpreferably, the amino acid sequence of the plasminogen of the presentinvention is as shown in SEQ ID No. 2, 6, 8, 10 or 12.

In one embodiment, the plasminogen is administered in combination with asuitable polypeptide carrier or stabilizer. In one embodiment, theplasminogen is administered at a dosage of 0.0001-2000 mg/kg, 0.001-800mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg/kg or 10-100mg/kg (by per kg of body weight) or 0.0001-2000 mg/cm², 0.001-800mg/cm², 0.01-600 mg/cm², 0.1-400 mg/cm², 1-200 mg/cm², 1-100 mg/cm² or10-100 mg/cm² (by per square centimeter of body surface area) daily,preferably the dosage is repeated at least once, preferably the dosageis administered at least daily. In the case of local administration, theabove dosages may also be further adjusted depending on thecircumstances.

In one embodiment, the plasminogen is administered by systemic ortopical route, preferably by the following routes: topical, intravenous,intramuscular, subcutaneous, inhalation, intraspinal, local injection,intraarticular injection or rectal route. In one embodiment, the localadministration is performed by applying a plasminogen-containingcatheter in the liver area.

In one aspect, the present invention relates to plasminogen or plasmin,a pharmaceutical composition comprising the plasminogen or plasmin, oran article or kit comprising the plasminogen or plasmin, which areuseful in the prevention and/or treatment of hepatic tissue injury andits related disorders in a subject.

In one embodiment, the hepatic tissue injury and its related disordersare hepatic injury and its related disorders caused by radiation orchemical substances. In one embodiment, the radiation or chemicalsubstances causing hepatic injury and its related disorders areradiotherapy or chemotherapy methods and drugs used for the treatment ofcancer. In one embodiment, the radiation is radiation caused by anaccident event. In one embodiment, the hepatic tissue injury and itsrelated disorders are toxic hepatic injury and its related disorders. Inone embodiment, the toxic hepatic injury is toxic hepatic injury causedby “hepatotropic poisons” including alcohol. In one embodiment, thehepatic tissue injury and its related disorders are caused by diabetesmellitus and one of the complications of diabetes mellitus. In oneembodiment, the hepatic tissue injury and its related disorders are dueto hepatitis caused by viral infection of the liver, such as hepatitiscaused by hepatitis A virus, hepatitis B virus, hepatitis C virus,hepatitis D virus or hepatitis E virus. In one embodiment, the hepatictissue injury and its related disorders are drug-induced hepatic injuryand its related disorders. In one embodiment, the hepatic tissue injuryand its related disorders are caused by intrahepatic blood congestion(liver congestion). In one embodiment, the hepatic tissue injury and itsrelated disorders are diabetic hepatic injury and its related disorders,toxic hepatic injury and its related disorders, drug-induced hepaticinjury or its related disorders, radiation-induced hepatic injury andits related disorders, viral infectious hepatic injury and its relateddisorders, or congestive hepatic injury and its related disorders. Inone embodiment, the hepatic tissue injury and its related disordersinclude hepatic dysfunction, abnormal hepatic enzymology, liverdiscomfort and haphalgesia, hepatomegaly, splenomegaly,hepatosplenomegaly, hepatitis, fatty liver, cholangitis, hepaticcirrhosis, hepatic necrosis and hepatic carcinoma caused by hepatictissue injury.

In one embodiment, the sequence in this patent application refers to thepatent document CN 102154253 A. In one embodiment, the plasminogen is aprotein that comprises a plasminogen active fragment and still hasplasminogen activity. In one embodiment, the plasminogen is selectedfrom Glu-plasminogen, Lys-plasminogen, mini-plasminogen,micro-plasminogen, δ-plasminogen or any combination thereof. In oneembodiment, the plasminogen or plasmin is administered systemically orlocally, including topical, intravenous, intramuscular, subcutaneous,inhalation, intraspinal, local injection, intraarticular injection orrectal administration. In one embodiment, the related disorders ofdiabetic hepatic injury or the related disorders of toxic hepatic injuryinclude: abnormal hepatic enzymology, liver discomfort and haphalgesia,hepatomegaly, splenomegaly, hepatosplenomegaly, hepatitis, fatty liver,cholangitis, hepatic cirrhosis, hepatic necrosis and hepatic carcinoma.In one embodiment, the hepatic injury and its related disorders arecaused by diabetes mellitus-induced angiopathy of large vessels, smallvessels, and microvessels. In one embodiment, the plasminogen can beadministered in combination with one or more other drugs. In oneembodiment, the other drugs include: liver-protecting drugs,antidiabetic drugs, antithrombotic drugs, anticoagulant drugs,hypolipidemic drugs, drugs against cardiovascular and cerebrovasculardiseases, and anti-infective drugs.

In one embodiment, the subject is a mammal, preferably human.

In one embodiment, the hepatic injury caused by diabetes mellitus iscaused by diabetes mellitus-induced angiopathy of large vessels, smallvessels, and microvessels.

In one embodiment, the subject has a low level of plasmin orplasminogen. Specifically, the low level is innate, secondary and/orlocal.

In one embodiment, the sequence in this patent application refers to thepatent document CN 102154253 A. In one embodiment, the plasminogen is aprotein that has 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35,1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2 or 1 amino acid added,deleted and/or substituted in SEQ ID No. 2, 6, 8, 10 or 12, and stillhas the activity of plasminogen. In one embodiment, the plasminogen is aprotein that comprises a plasminogen active fragment and still has theactivity of plasminogen. In one embodiment, the plasminogen is selectedfrom Glu-plasminogen, Lys-plasminogen, mini-plasminogen,micro-plasminogen, δ-plasminogen or any combination thereof. In oneembodiment, the plasminogen is a conservatively substituted variantselected from variants of Glu-plasminogen, Lys-plasminogen,mini-plasminogen, δ-plasminogen or micro-plasminogen. In one embodiment,the plasminogen is a human natural plasminogen, such as an ortholog ofplasminogen shown in SEQ ID No. 2, e.g., an ortholog of plasminogen fromprimates or rodents, for example, an ortholog of plasminogen fromgorillas, rhesus monkeys, murine, cows, horses and dogs. Mostpreferably, the amino acid sequence of the plasminogen of the presentinvention is as shown in SEQ ID No. 2, 6, 8, 10 or 12.

In one embodiment, the plasminogen is administered in combination with asuitable polypeptide carrier or stabilizer. In one embodiment, theplasminogen is administered at a dosage of 0.0001-2000 mg/kg, 0.001-800mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg/kg or 10-100mg/kg (by per kg of body weight) or 0.0001-2000 mg/cm², 0.001-800mg/cm², 0.01-600 mg/cm², 0.1-400 mg/cm², 1-200 mg/cm², 1-100 mg/cm² or10-100 mg/cm² (by per square centimeter of body surface area) daily,preferably the dosage is repeated at least once, preferably the dosageis administered at least daily. In the case of local administration, theabove dosages may also be further adjusted depending on thecircumstances.

In one embodiment, the plasminogen is administered by systemic ortopical route, preferably by the following routes: topical, intravenous,intramuscular, subcutaneous, inhalation, intraspinal, local injection,intraarticular injection or rectal route. In one embodiment, the localadministration is performed by applying a plasminogen-containingcatheter in the liver area.

In one embodiment, the plasminogen or plasmin is subpackaged incontainers. Preferably, the article or kit further comprises other drugssubpackaged in other containers of the kit. The kit can also compriseinstructions for use, which indicate that the plasminogen can be used totreat hepatic tissue injury and its related disorders, specifically, forexample, diabetic hepatic injury and its related disorders caused bydiabetes mellitus, toxic hepatic injury and its related disorders,drug-induced hepatic injury or its related disorders, hepatic injury andits related disorders caused by radiation, hepatic injury and itsrelated disorders caused by virus infection, or hepatic injury and itsrelated disorders caused by congestion, and can further indicate thatthe plasminogen or plasmin can be administered before, simultaneouslywith and/or after administration of other drugs or therapies.

The present invention explicitly encompasses all the combinations oftechnical features belonging to the embodiments of the presentinvention, and these combined technical solutions have been explicitlydisclosed in the present application, as if the above technicalsolutions were individually and explicitly disclosed. In addition, thepresent invention also explicitly encompasses all the subcombinations ofthe various embodiments and elements thereof, and these subcombinationshave been disclosed herein, as if each of such subcombinations wasindividually and explicitly disclosed herein.

DETAILED DESCRIPTION OF EMBODIMENTS

“Diabetic hepatic injury” refers to a lesion with the histological andfunctional changes of the liver caused by diabetes mellitus. It ismainly caused by diabetes mellitus-induced angiopathy of large vessels,small vessels, and microvessels. Hepatic injuries known to be caused bydiabetes mellitus include: hepatic enzymology abnormalities, which cancause carbon dioxide accumulation, acidosis, reduced oxygen supply andincreased oxygen consumption in hepatocytes, resulting in increasedactivity of liver transaminases and bilirubin metabolism disorder, withsevere cases causing hepatocyte necrosis; fatty liver, wherein diabetesmellitus is the third most common cause of fatty liver among all causesof fatty liver, and 21%-78% of diabetics have fatty liver; andhepatitis, cirrhosis and hepatic carcinoma, wherein the prevalence ofviral hepatitis in diabetics is about 2-4 times that in normal people,and the incidence of primary hepatic carcinoma is about 4 times that innormal people.

“Chemical hepatic injury” or “toxic hepatic injury” refers to hepaticdamage caused by chemical hepatotoxic substances. These chemicalsubstances include alcohol, chemical toxicants in the environment andcertain drugs. There are substances that are toxic to the liver in bothnature and human industrial production processes, known as “hepatotropicpoisons”. People are generally susceptible to these poisons, and theyhave a short incubation period. The pathological process is directlyrelated to the amount of chemical substances, and these substances maycause different levels of hepatocyte necrosis, steatosis and hepaticcirrhosis in the liver.

“hepatotropic poisons” refer to a generic term for substances that aretoxic to the liver. Alcohol is the most common “hepatotropic poison” inlife. In addition to alcohol, chemical toxicants in the environment andcertain drugs may also cause hepatic injury. As an importantdetoxification organ for the human body, the liver has dual blood supplyfrom the hepatic arteries and hepatic veins. Chemical substances canenter the liver through the portal veins of the gastrointestinal tractor the systemic circulation for conversion, and thus the liver isvulnerable to toxic substances in chemicals. There are substances thatare toxic to the liver in both nature and human industrial productionprocesses, known as “hepatotropic poisons”. They enter the liver and maycause different levels of hepatocyte necrosis, steatosis and hepaticcirrhosis in the liver. The pathological manifestations include (1)steatosis. Carbon tetrachloride, yellow phosphorus and the like caninterfere with the synthesis and translocation of lipoproteins, formingfatty liver. (2) Lipid peroxidation, which is a special manifestationform of toxic hepatic injury. For example, carbon tetrachloride ismetabolized in vivo to produce an intermediate product with strongoxidizing ability, leading to lipid peroxidation on biofilms,destructing membrane phospholipids and changing the structures andfunctions of cells. (3) Cholestasis, which is mainly associated withdamage of the hepatic cell membrane and microvilli, causing bile acidexcretion disorder.

“Drug-induced hepatic injury”, also known as drug-induced hepatopathy,refers to hepatic injury during drug use caused by the drug itselfor/and its metabolites, or the hypersensitivity or reduced tolerance tothe drug due to a special constitution of the body, clinicallymanifested as a variety of acute and chronic hepatopathies. Patientswith light illness may recover spontaneously after discontinuation ofmedication, and serious ones may be life-threatening and require activetreatment and rescue. “Drug-induced hepatic injury” can occur in ahealthy person who has not previously had a history of hepatopathy or apatient who has had a serious disease previously. It can occur when themedication is overdosed, but it can also occur under normal dosages.

“Radiation-induced hepatic injury” refers to radiation injury caused byhigh-energy ionizing radiation, including α and β particles, γ rays, χrays and neutron rays. Either instant irradiation with high-dose rays orprolonged irradiation with low-dose rays may cause tissue injury. Theradiation energy destroys the chromosomes and enzymes of cells anddisrupts the normal functions of cells.

“Viral infectious hepatic injury” refers to a generic term for hepaticinjury caused by viral infection. The viral infection commonly includesinfection caused by hepatitis A virus, hepatitis B virus, hepatitis Cvirus, hepatitis D virus or hepatitis E virus.

“Congestive hepatic injury” refers to pathology of hepatic tissue injurycaused by intrahepatic blood congestion. Any disease that blocks thereturn of inferior vena cava blood to the heart can lead to livercongestion, such as rheumatic valvular heart disease, chronicconstrictive pericarditis, hypertensive heart disease, ischemic heartdisease, pulmonary heart disease and congenital heart disease.

“Plasminogen” is the zymogenic form of plasmin, and based on thesequence in the swiss prot and calculated from the amino acid sequence(SEQ ID No. 4) of the natural human-derived plasminogen containing asignal peptide, is a glycoprotein composed of 810 amino acids, which hasa molecular weight of about 92 kD and is synthesized mainly in the liverand capable of circulating in the blood; and the cDNA sequence encodingthis amino acid sequence is as shown in SEQ ID No3. Full-lengthplasminogen contains seven domains: a C-terminal serine protease domain,an N-terminal Pan Apple (PAp) domain and five Kringle domains (Kringles1-5). Referring to the sequence in the swiss prot, the signal peptidecomprises residues Met1-Gly19, PAp comprises residues Glu20-Va198,Kringle 1 comprises residues Cys103-Cys181, Kringle 2 comprises residuesGlu184-Cys262, Kringle 3 comprises residues Cys275-Cys352, Kringle 4comprises residues Cys377-Cys454, and Kringle 5 comprises residuesCys481-Cys560. According to the NCBI data, the serine protease domaincomprises residues Va1581-Arg804.

Glu-plasminogen is a natural full-length plasminogen and is composed of791 amino acids (without a signal peptide of 19 amino acids); the cDNAsequence encoding this sequence is as shown in sequence 1; and the aminoacid sequence is as shown in SEQ ID No. 2. In vivo, Lys-plasminogen,which is formed by hydrolysis of amino acids at positions 76-77 ofGlu-plasminogen, is also present, as shown in SEQ ID No. 6; and the cDNAsequence encoding this amino acid sequence is as shown in SEQ ID No. 5.δ-plasminogen is a fragment of full-length plasminogen that lacks thestructure of Kringle 2-Kringle 5 and contains only Kringle 1 and theserine protease domain^([8,9]). The amino acid sequence (SEQ ID No. 8)of δ-plasminogen has been reported in the literature^([9]), and the cDNAsequence encoding this amino acid sequence is as shown in SEQ ID No. 7.Mini-plasminogen is composed of Kringle 5 and the serine proteasedomain, and has been reported in the literature to comprise residuesVa1443-Asn791 (with the Glu residue of the Glu-plasminogen sequence thatdoes not contain a signal peptide as the starting amino acid)^([10]);the amino acid sequence is as shown in SEQ ID No. 10; and the cDNAsequence encoding this amino acid sequence is as shown in SEQ ID No. 9.Micro-plasminogen comprises only the serine protease domain, the aminoacid sequence of which has been reported in the literature to compriseresidues Ala543-Asn791 (with the Glu residue of the Glu-plasminogensequence that does not contain a signal peptide as the starting aminoacid)^([11]), and the sequence of which has been also reported in patentdocument CN 102154253 A to comprise residues Lys531-Asn791 (with the Gluresidue of the Glu-plasminogen sequence that does not contain a signalpeptide as the starting amino acid) (the sequence in this patentapplication refers to the patent document CN 102154253 A); the aminoacid sequence is as shown in SEQ ID No. 12; and the cDNA sequenceencoding this amino acid sequence is as shown in SEQ ID No. 11.

In the present invention, “plasmin” is used interchangeably with“fibrinolysin” and “fibrinoclase”, and the terms have the same meaning;and “plasminogen” is used interchangeably with “fibrinolytic zymogen”and “fibrinoclase zymogen”, and the terms have the same meaning.

Those skilled in the art can understand that all the technical solutionsof the plasminogen of the present invention are suitable for plasmin.Therefore, the technical solutions described in the present inventioncover plasminogen and plasmin.

In the course of circulation, plasminogen is in a closed, inactiveconformation, but when bound to thrombi or cell surfaces, it isconverted into an active plasmin in an open conformation under themediation of a plasminogen activator (PA). The active plasmin canfurther hydrolyze the fibrin clots to fibrin degradation products andD-dimers, thereby dissolving the thrombi. The PAp domain of plasminogencomprises an important determinant that maintains plasminogen in aninactive, closed conformation, and the KR domain is capable of bindingto lysine residues present on receptors and substrates. A variety ofenzymes that can serve as plasminogen activators are known, including:tissue plasminogen activator (tPA), urokinase plasminogen activator(uPA), kallikrein, coagulation factor XII (Hagmann factor), and thelike.

“Plasminogen active fragment” refers to an active fragment in theplasminogen protein that is capable of binding to a target sequence in asubstrate and exerting the proteolytic function. The technical solutionsof the present invention involving plasminogen encompass technicalsolutions in which plasminogen is replaced with a plasminogen activefragment. The plasminogen active fragment of the present invention is aprotein comprising a serine protease domain of plasminogen. Preferably,the plasminogen active fragment of the present invention comprises SEQID NO: 14, or an amino acid sequence having an amino acid sequenceidentity of at least 80%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO:14. Therefore, plasminogen of the present invention comprises a proteincomprising the plasminogen active fragment and still having plasminogenactivity.

At present, methods for determining plasminogen and its activity inblood include: detection of tissue plasminogen activator activity(t-PAA), detection of tissue plasminogen activator antigen (t-PAAg) inplasma, detection of tissue plasminogen activity (plgA) in plasma,detection of tissue plasminogen antigen (plgAg) in plasma, detection ofactivity of the inhibitor of tissue plasminogen activators in plasma,detection of inhibitor antigens of tissue plasminogen activators inplasma and detection of plasmin-anti-plasmin (PAP) complex in plasma.The most commonly used detection method is the chromogenic substratemethod: streptokinase (SK) and a chromogenic substrate are added to atest plasma, the PLG in the test plasma is converted into PLM by theaction of SK, PLM acts on the chromogenic substrate, and then it isdetermined that the increase in absorbance is directly proportional toplasminogen activity using a spectrophotometer. In addition, plasminogenactivity in blood can also be determined by immunochemistry, gelelectrophoresis, immunonephelometry, radioimmuno-diffusion and the like.

“Orthologues or orthologs” refer to homologs between different species,including both protein homologs and DNA homologs, and are also known asorthologous homologs and vertical homologs. The term specifically refersto proteins or genes that have evolved from the same ancestral gene indifferent species. The plasminogen of the present invention includeshuman natural plasminogen, and also includes orthologues or orthologs ofplasminogens derived from different species and having plasminogenactivity.

“Conservatively substituted variant” refers to one in which a givenamino acid residue is changed without altering the overall conformationand function of the protein or enzyme, including, but not limited to,replacing an amino acid in the amino acid sequence of the parent proteinby an amino acid with similar properties (such as acidity, alkalinityand hydrophobicity). Amino acids with similar properties are well known.For example, arginine, histidine and lysine are hydrophilic basic aminoacids and are interchangeable. Similarly, isoleucine is a hydrophobicamino acid that can be replaced by leucine, methionine or valine.Therefore, the similarity of two proteins or amino acid sequences withsimilar functions may be different. For example, the similarity(identity) is 70%-99% based on the MEGALIGN algorithm. “Conservativelysubstituted variant” also includes a polypeptide or enzyme having aminoacid identity of 60% or more, preferably 75% or more, more preferably85% or more, even more preferably 90% or more as determined by the BLASTor FASTA algorithm, and having the same or substantially similarproperties or functions as the natural or parent protein or enzyme.

“Isolated” plasminogen refers to the plasminogen protein that isisolated and/or recovered from its natural environment. In someembodiments, the plasminogen will be purified (1) to a purity of greaterthan 90%, greater than 95% or greater than 98% (by weight), asdetermined by the Lowry method, such as more than 99% (by weight); (2)to a degree sufficiently to obtain at least 15 residues of theN-terminal or internal amino acid sequence using a spinning cupsequenator; or (3) to homogeneity, which is determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing ornon-reducing conditions using Coomassie blue or silver staining.Isolated plasminogen also includes plasminogen prepared from recombinantcells by bioengineering techniques and separated by at least onepurification step.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein and refer to polymeric forms of amino acids ofany length, which may include genetically encoded and non-geneticallyencoded amino acids, chemically or biochemically modified or derivatizedamino acids, and polypeptides having modified peptide backbones. Theterm includes fusion proteins, including, but not limited to, fusionproteins having heterologous amino acid sequences, fusions havingheterologous and homologous leader sequences (with or without N-terminalmethionine residues), and the like.

The “percent amino acid sequence identity (%)” with respect to thereference polypeptide sequence is defined as the percentage of aminoacid residues in the candidate sequence identical to the amino acidresidues in the reference polypeptide sequence when a gap is introducedas necessary to achieve maximal percent sequence identity and noconservative substitutions are considered as part of sequence identity.The comparison for purposes of determining percent amino acid sequenceidentity can be achieved in a variety of ways within the skill in theart, for example using publicly available computer softwares, such asBLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for aligning sequences,including any algorithm needed to achieve the maximum comparison overthe full length of the sequences being compared. However, for purposesof the present invention, the percent amino acid sequence identity valueis generated using the sequence comparison computer program ALIGN-2.

In the case of comparing amino acid sequences using ALIGN-2, the % aminoacid sequence identity of a given amino acid sequence A relative to agiven amino acid sequence B (or may be expressed as a given amino acidsequence A having or containing a certain % amino acid sequence identityrelative to, with or for a given amino acid sequence B) is calculated asfollows:

fraction X/Y×100

wherein X is the number of identically matched amino acid residuesscored by the sequence alignment program ALIGN-2 in the alignment of Aand B using the program, and wherein Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A relative to B will not be equal tothe % amino acid sequence identity of B relative to A. Unlessspecifically stated otherwise, all the % amino acid sequence identityvalues used herein are obtained using the ALIGN-2 computer program asdescribed in the previous paragraph.

As used herein, the terms “treatment” and “treating” refer to obtaininga desired pharmacological and/or physiologic effect. The effect may becomplete or partial prevention of a disease or its symptoms and/orpartial or complete cure of the disease and/or its symptoms, andincludes: (a) prevention of the disease from developing in a subjectthat may have a predisposition to the disease but has not been diagnosedas having the disease; (b) suppression of the disease, i.e., blockingits formation; and (c) alleviation of the disease and/or its symptoms,i.e., eliminating the disease and/or its symptoms.

The terms “individual”, “subject” and “patient” are used interchangeablyherein and refer to mammals, including, but not limited to, murine (ratsand mice), non-human primates, humans, dogs, cats, hoofed animals (e.g.,horses, cattle, sheep, pigs, goats) and so on.

“Therapeutically effective amount” or “effective amount” refers to anamount of plasminogen sufficient to achieve the prevention and/ortreatment of a disease when administered to a mammal or another subjectto treat the disease. The “therapeutically effective amount” will varydepending on the plasminogen used, the severity of the disease and/orits symptoms, as well as the age, body weight of the subject to betreated, and the like.

Preparation of the Plasminogen of the Present Invention

Plasminogen can be isolated and purified from nature for furthertherapeutic uses, and can also be synthesized by standard chemicalpeptide synthesis techniques. When chemically synthesized, a polypeptidecan be subjected to liquid or solid phase synthesis. Solid phasepolypeptide synthesis (SPPS) is a method suitable for chemical synthesisof plasminogen, in which the C-terminal amino acid of a sequence isattached to an insoluble support, followed by the sequential addition ofthe remaining amino acids in the sequence. Various forms of SPPS, suchas Fmoc and Boc, can be used to synthesize plasminogen. Techniques forsolid phase synthesis are described in Barany and Solid-Phase PeptideSynthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol.2: Special Methods in Peptide Synthesis, Part A., Merrifield et al. J.Am. Chem. Soc., 85: 2149-2156 (1963); Stewart et al. Solid Phase PeptideSynthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); and GanesanA. 2006 Mini Rev. Med Chem. 6:3-10 and Camarero J A et al. 2005 ProteinPept Lett. 12:723-8. Briefly, small insoluble porous beads are treatedwith a functional unit on which a peptide chain is constructed. Afterrepeated cycles of coupling/deprotection, the attached solid phase freeN-terminal amine is coupled to a single N-protected amino acid unit.This unit is then deprotected to expose a new N-terminal amine that canbe attached to another amino acid. The peptide remains immobilized onthe solid phase before it is cut off.

Standard recombinant methods can be used to produce the plasminogen ofthe present invention. For example, a nucleic acid encoding plasminogenis inserted into an expression vector, so that it is operably linked toa regulatory sequence in the expression vector. Expression regulatorysequence includes, but is not limited to, promoters (e.g., naturallyassociated or heterologous promoters), signal sequences, enhancerelements and transcription termination sequences. Expression regulationcan be a eukaryotic promoter system in a vector that is capable oftransforming or transfecting eukaryotic host cells (e.g., COS or CHOcells). Once the vector is incorporated into a suitable host, the hostis maintained under conditions suitable for high-level expression of thenucleotide sequence and collection and purification of plasminogen.

A suitable expression vector is usually replicated in a host organism asan episome or as an integral part of the host chromosomal DNA. Ingeneral, an expression vector contains a selective marker (e.g.,ampicillin resistance, hygromycin resistance, tetracycline resistance,kanamycin resistance or neomycin resistance) to facilitate detection ofthose exogenous cells transformed with a desired DNA sequence.

Escherichia coli is an example of prokaryotic host cells that can beused to clone a polynucleotide encoding the subject antibody. Othermicrobial hosts suitable for use include Bacillus, for example, Bacillussubtilis and other species of Enterobacteriaceae (such as Salmonellaspp. and Serratia spp.), and various Pseudomonas spp. In theseprokaryotic hosts, expression vectors can also be generated which willtypically contain an expression control sequence (e.g., origin ofreplication) that is compatible with the host cell. In addition, therewill be many well-known promoters, such as the lactose promoter system,the tryptophan (trp) promoter system, the beta-lactamase promoter systemor the promoter system from phage lambda. Optionally in the case ofmanipulation of a gene sequence, a promoter will usually controlexpression, and has a ribosome binding site sequence and the like toinitiate and complete transcription and translation.

Other microorganisms, such as yeast, can also be used for expression.Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitableyeast host cells, in which a suitable vector has an expression controlsequence (e.g., promoter), an origin of replication, a terminationsequence and the like, as required. A typical promoter comprises3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeastpromoters specifically include promoters derived from alcoholdehydrogenase, isocytochrome C, and enzymes responsible for maltose andgalactose utilization.

In addition to microorganisms, mammalian cells (eg, mammalian cellscultured in in vitro cell culture) can also be used to express andproduce the protein of the invention (eg, polynucleotides encoding thesubject protein). See Winnacker, From Genes to Clones, VCH Publishers,N.Y., N.Y. (1987). Suitable mammalian host cells include CHO cell lines,various Cos cell lines, HeLa cells, myeloma cell lines and transformed Bcells or hybridomas. Expression vectors for these cells may comprise anexpression control sequence, such as an origin of replication, promoterand enhancer (Queen et al. Immunol. Rev. 89:49 (1986)), as well asnecessary processing information sites, such as a ribosome binding site,RNA splice site, polyadenylation site and transcription terminatorsequence. Examples of suitable expression control sequences arepromoters derived from white immunoglobulin gene, SV40, adenovirus,bovine papilloma virus, cytomegalovirus and the like. See Co et al. J.Immunol. 148:1149 (1992).

Once synthesized (chemically or recombinantly), the plasminogen of thepresent invention can be purified according to standard procedures inthe art, including ammonium sulfate precipitation, affinity column,column chromatography, high performance liquid chromatography (HPLC),gel electrophoresis and the like. The plasminogen is substantially pure,e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, atleast about 90% to 95% pure, or 98% to 99% pure or purer, for examplefree of contaminants such as cell debris, macromolecules other than theplasminogen and the like.

Pharmaceutical Formulations

A therapeutic formulation can be prepared by mixing plasminogen of adesired purity with an optional pharmaceutical carrier, excipient orstabilizer (Remington's Pharmaceutical Sciences, 16th edition, Osol, A.ed. (1980)) to form a lyophilized preparation or an aqueous solution.Acceptable carriers, excipients and stabilizers are non-toxic to therecipient at the dosages and concentrations employed, and includebuffers, such as phosphates, citrates and other organic acids;antioxidants, including ascorbic acid and methionine; preservatives(e.g., octadecyl dimethyl benzyl ammonium chloride; hexane chloridediamine; benzalkonium chloride and benzethonium chloride; phenol,butanol or benzyl alcohol; alkyl p-hydroxybenzoates, such as methyl orpropyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol;3-pentanol; and m-cresol); low molecular weight polypeptides (less thanabout 10 residues); proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone;amino acids, such as glycine, glutamine, asparagine, histidine, arginineor lysine; monosaccharides, disaccharides and other carbohydrates,including glucose, mannose or dextrins; chelating agents, such as EDTA;sugars, such as sucrose, mannitol, fucose or sorbitol; salt-formingcounterions, such as sodium; metal complexes (e.g., zinc-proteincomplexes); and/or non-ionic surfactants, such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulations of the invention may also comprise one or more activecompounds required for the particular disorder to be treated, preferablythose that are complementary in activity and have no side effects withone another, for example anti-hypertensive drugs, anti-arrhythmic drugs,drugs for treating diabetes mellitus, and the like.

The plasminogen of the present invention may be encapsulated inmicrocapsules prepared by techniques such as coacervation or interfacialpolymerization, for example, it may be incorporated in a colloid drugdelivery system (e.g., liposomes, albumin microspheres, microemulsions,nanoparticles and nanocapsules), or incorporated inhydroxymethylcellulose or gel-microcapsules and poly-(methylmethacrylate) microcapsules in macroemulsions. These techniques aredisclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A.Ed. (1980).

The plasminogen of the present invention for in vivo administration mustbe sterile. This can be easily achieved by filtration through a sterilefiltration membrane before or after freeze drying and reconstitution.

The plasminogen of the present invention can be prepared into asustained-release preparation. Suitable examples of sustained-releasepreparations include solid hydrophobic polymer semi-permeable matriceshaving a shape and containing glycoproteins, such as films ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate)) (Langeret al. J. Biomed. Mater. Res., 15: 167-277 (1981); and Langer, Chem.Tech., 12:98-105 (1982)), or poly(vinyl alcohol), polylactides (U.S.Pat. No. 3,773,919, and EP 58, 481), copolymer of L-glutamic acid and γethyl-L-glutamic acid (Sidman et al. Biopolymers 22:547 (1983)),nondegradable ethylene-vinyl acetate (Langer et al. supra), ordegradable lactic acid-glycolic acid copolymers such as Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly D-(−)-3-hydroxybutyric acid. Polymers,such as ethylene-vinyl acetate and lactic acid-glycolic acid, are ableto persistently release molecules for 100 days or longer, while somehydrogels release proteins for a shorter period of time. A rationalstrategy for protein stabilization can be designed based on relevantmechanisms. For example, if the aggregation mechanism is discovered tobe formation of an intermolecular S—S bond through thio-disulfideinterchange, stability is achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

Administration and Dosage

The pharmaceutical composition of the present invention can beadministered in different ways, for example by intravenous,intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial(e.g., via carotid), intramuscular, intranasal, topical or intradermaladministration or spinal cord or brain delivery. An aerosol preparation,such as a nasal spray preparation, comprises purified aqueous or othersolutions of the active agent along with a preservative and isotonicagent. Such preparations are adjusted to a pH and isotonic statecompatible with the nasal mucosa.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, and alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, or fixed oils. Intravenousvehicles include liquid and nutrient supplements, electrolytesupplements and the like. Preservatives and other additives may also bepresent, for example, such as antimicrobial agents, antioxidants,chelating agents and inert gases.

The medical staff will determine the dosage regimen based on variousclinical factors. As is well known in the medical field, the dosage ofany patient depends on a variety of factors, including the patient'ssize, body surface area, age, the specific compound to be administered,sex, frequency and route of administration, overall health and otherdrugs administered simultaneously. The dosage range of thepharmaceutical composition comprising plasminogen of the presentinvention may be, for example, about 0.0001 to 2000 mg/kg, or about0.001 to 500 mg/kg (such as 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75mg/kg, 10 mg/kg and 50 mg/kg) of the subject's body weight daily. Forexample, the dosage may be 1 mg/kg body weight or 50 mg/kg body weight,or in the range of 1 mg/kg-50 mg/kg, or at least 1 mg/kg. Dosages aboveor below this exemplary range are also contemplated, especiallyconsidering the above factors. The intermediate dosages in the aboverange are also included in the scope of the present invention. A subjectmay be administered with such dosages daily, every other day, weekly orbased on any other schedule determined by empirical analysis. Anexemplary dosage schedule includes 1-10 mg/kg for consecutive days.During administration of the drug of the present invention, thetherapeutic effect and safety of diabetic hepatopathy and its relateddisorders are required to be assessed real-timely and regularly.

Treatment Efficacy and Treatment Safety

One embodiment of the present invention relates to the judgment oftreatment efficacy and treatment safety after treating a subject withplasminogen. The methods for judging the treatment efficacy include, butare not limited to: 1) examining the liver function of a subject, forexample, enzymatic levels in the patient, such as levels of serumaspartate aminotransferase (AST), alanine transaminase (ALT), totalbilirubin, direct bilirubin, indirect bilirubin, albumin, globulin,cholinesterase, alkaline phosphatase and transpeptidase, are examined todetermine whether they are in the normal value ranges or not, and aftertreating the subject with plasminogen of the present invention, it isexpected that the above liver function indexes will return to normalvalue or be improved, such as alanine transaminase (ALT) at 0-40μ/L,aspartate transaminase (AST) at 0-40 μL, gamma glutamyl transferase(GGT) of less than 40 units and total bilirubin at 3.4-20.5 μmol/L; 2)examining the prothrombin time (PT) and prothrombin activity (PTA) ofthe subject: PT is an important index reflecting the function of hepaticcoagulation factor synthesis; PTA is a commonly used expression methodfor PT measurement value and of great value in judging the progressionand prognosis of hepatopathy, in which the progressive decrease of PTAto less than 40% is an important diagnostic criterion for hepaticfailure, and to less than 20% indicates hepatic dysfunction; and aftertreating the subject with plasminogen and its variants of the presentinvention, the decrease in PTA of the patient is expected to beremarkably improved; 3) imageological examination: including abdominalcolor ultrasonography of the liver, gallbladder and spleen, CT or MRI tolearn the degree of recovery of hepatic injury; 4) examining tumormarkers, such as alpha fetal protein (AFP), CA199 and AFU; and 5)hepatic biopsy to determine the degree of recovery of fibrosis and otherinjuries. In addition, the present invention also relates to thejudgment of the safety of the therapeutic regimen during and aftertreating a subject with plasminogen and its variants, including, but notlimited to, statistics of serum half-life, half-life of treatment,median toxic dose (TD50) and median lethal dose (LD50) in the subject,or observing various adverse events such as sensitization that occurduring or after treatment.

Articles or Kits

One embodiment of the present invention relates to an article or kitcomprising plasminogen of the present invention useful in the treatmentof the hepatic injury and its related disorders caused by diabetesmellitus. The article preferably includes a container, label or packageinsert. Suitable containers include bottles, vials, syringes and thelike. The container can be made of various materials, such as glass orplastic. The container contains a composition that is effective to treatthe disease or disorder of the present invention and has a sterileaccess (for example, the container may be an intravenous solution bag orvial containing a plug that can be pierced by a hypodermic injectionneedle). At least one active agent in the composition isplasminogen/plasmin. The label on or attached to the container indicatesthat the composition is used to treat the hepatic injury and its relateddisorders caused by diabetes mellitus according to the presentinvention. The article may further comprise a second containercontaining a pharmaceutically acceptable buffer, such as phosphatebuffered saline, Ringer's solution and glucose solution. It may furthercomprise other substances required from a commercial and userperspective, including other buffers, diluents, filters, needles andsyringes. In addition, the article comprises a package insert withinstructions for use, including, for example, instructions to a user ofthe composition to administer the plasminogen composition and otherdrugs to treat an accompanying disease to a patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows changes in body weight after administration of plasminogento 24-25-week-old diabetic mice.

FIG. 2 shows the observed results of HE staining of the liver afteradministration of plasminogen to 24-25-week-old diabetic mice for 15consecutive days.

FIG. 3 shows the observed results of fibrin immunostaining of the liverunder a microscopy after administration of plasminogen to 24-25-week-olddiabetic mice for 15 consecutive days.

FIG. 4 shows changes in body weight after administration of plasminogento 24-25-week-old diabetic mice for 31 consecutive days.

FIG. 5 shows the observed results of HE staining of the liver afteradministration of plasminogen to 24-25-week-old diabetic mice for 31consecutive days.

FIG. 6 shows the observed results of fibrin immunostaining of the liverafter administration of plasminogen to 24-25-week-old diabetic mice for31 consecutive days.

FIG. 7 shows the observed results of F4/80 immunostaining of the liverafter administration of plasminogen to 24-25-week-old diabetic mice for31 consecutive days.

FIG. 8 shows the detection results of alanine transaminase (ALT) inserum after administration of PBS or plasminogen to 24-25-week-olddiabetic mice for 31 days.

FIG. 9 shows the observed results of HE staining of the liver on days 0,2 and 7 after administration of plasminogen to mice with acute hepaticinjury induced by carbon tetrachloride.

FIG. 10 shows the observed results of HE staining of the liver at hours18, 24 and 48 after administration of plasminogen to plg^(−/−) mice withacute hepatic injury induced by carbon tetrachloride.

FIG. 11 shows the observed results of fibrin immunostaining of the liverat hours 18, 24 and 48 after administration of plasminogen to pig^(−/−)mice with acute hepatic injury induced by carbon tetrachloride.

FIG. 12 shows the observed results of F4/80 immunostaining of the liver10 days after administration of plasminogen to mice irradiated with 5.0Gy X-rays.

FIG. 13 shows the observed results of fibrin immunostaining of the liver7 days after administration of plasminogen to model mice with injuryinduced by 10 mg/Kg cisplatin chemotherapy.

FIG. 14 shows the observed results of HE staining of the liver at hours18, 24 and 48 and 7 days after administration of plasminogen toplg^(−/−) mice with acute hepatic injury induced by carbontetrachloride.

EXAMPLES Example 1. Effect of Plasminogen on Body Weight of LateDiabetic Mice with Nerve Injury

Ten male db/db mice aged 24-25 weeks were randomly divided into twogroups, five in the control group administered with vehicle PBS and fivein the group administered with plasminogen, respectively. The day whenthe experiment began was recorded on Day 0, and the mice were weighedand grouped. From the second day of the experiment, plasminogen or PBSwas administered to the mice for 15 consecutive days, and the day wasrecorded as Day 1. Mice in the group administered with plasminogen wereinjected with plasminogen at a dose of 2 mg/0.2 mL/mouse/day via thetail vein, and an equal volume of PBS was administered to mice in thecontrol group administered with vehicle PBS. The mice were weighed ondays 0, 4, 7, 11 and 16 after administration of plasminogen,respectively. The results showed that there was no significantdifference in body weight between mice in the group administered withplasminogen and those in the control group administered with vehicle PBSon days 0, 4, 7, 11 and 16 (FIG. 1), indicating that plasminogen haslittle effect on animal body weight.

Example 2. Protective Effect of Plasminogen on Late Hepatic TissueInjury of Mice with Diabetic Hepatic Injury

Ten male db/db mice aged 24-25 weeks were randomly divided into twogroups, five in the control group administered with vehicle PBS and fivein the group administered with plasminogen, respectively. The day whenthe experiment began was recorded on Day 0, and the mice were weighedand grouped. From the second day of the experiment, plasminogen or PBSwas administered to the mice for 15 consecutive days, and the day wasrecorded as Day 1. Mice in the group administered with plasminogen wereinjected with plasminogen at a dose of 2 mg/0.2 mL/mouse/day via thetail vein, and an equal volume of PBS was administered to mice in thecontrol group administered with vehicle PBS. Mice were sacrificed on day16, and liver tissues were fixed in 10% neutral formalin fix solutionfor 24-48 hours. The fixed liver tissues were paraffin-embedded afterdehydration with alcohol gradient and permeabilization with xylene. Thethickness of the tissue sections was 5 μm. The sections were dewaxed andrehydrated, stained with hematoxylin and eosin (HE staining),differentiated with 1% hydrochloric acid in alcohol, and returned toblue with ammonia water. The sections were sealed after dehydration withalcohol gradient.

The HE staining results showed that in mice in the control groupadministered with vehicle PBS, the hepatocytes showed severe steatosisand lipid deposition, cell nuclei were squeezed to the edge, the cellsshowed mild hydropic degeneration, and the hepatic cord was disordered;and compared with mice in the control group administered with vehicle,in mice in the group administered with plasminogen, hepatocytes showedrelieved steatosis, mild steatosis and mainly moderate hydropicdegeneration. This indicated that plasminogen can promote the repair ofdiabetic hepatic injury.

Example 3. Plasminogen Reduces the Fibrin Level in Liver Tissues ofDiabetic Mice

Ten male db/db mice aged 24-25 weeks were randomly divided into twogroups, five in the control group administered with vehicle PBS and fivein the group administered with plasminogen, respectively. The day whenthe experiment began was recorded on Day 0, and the mice were weighedand grouped. From the second day of the experiment, plasminogen or PBSwas administered to the mice for 15 consecutive days, and the day wasrecorded as Day 1. Mice in the group administered with plasminogen wereinjected with plasminogen at a dose of 2 mg/0.2 mL/mouse/day via thetail vein, and an equal volume of PBS was administered to mice in thecontrol group administered with vehicle PBS. Mice were sacrificed on day16, and liver tissues were fixed in 10% neutral formalin fix solutionfor 24-48 hours. The fixed liver tissues were paraffin-embedded afterdehydration with alcohol gradient and permeabilization with xylene. Thethickness of the tissue sections was 5 μm. The sections were dewaxed andrehydrated and washed with water once. The sections were incubated with3% hydrogen peroxide for 15 minutes and wash with water twice for 5minutes each time. The sections were blocked with 10% normal goat serumsolution (Vector laboratories, Inc., USA) for 1 hour; and after the timewas up, the goat serum solution was discarded, and the tissues werecircled with a PAP pen. The sections were incubated with rabbitanti-mouse fibrin (fibrinogen) antibody (Abcam) overnight at 4° C. andwashed with TBS twice for 5 minutes each time. The sections wereincubated with a secondary antibody, goat anti-rabbit IgG (HRP) antibody(Abcam), for 1 hour at room temperature and washed with TBS twice for 5minutes each time. The sections were developed with a DAB kit (Vectorlaboratories, Inc., USA). After washing with water three times, thesections were counterstained with hematoxylin for 30 seconds, flushedwith running water for 5 minutes, and then washed with TBS once. Aftergradient dehydration, permeabilization and sealing, the sections wereobserved under a microscope at 200×.

Fibrinogen is the precursor of fibrin, and in the presence of tissueinjury, as a stress response to the body's injury, fibrinogen ishydrolyzed into fibrin^([1244]). Therefore, the fibrinogen level can beused as a sign of the degree of injury.

The study found that compared with mice in the control groupadministered with vehicle PBS (FIG. 3A), those in the group administeredwith plasminogen (FIG. 3B) had a decreased fibrin level in the livertissues, indicating that plasminogen has the function of inhibiting thefibrin deposition and the injury is repaired to a certain degree.

Example 4. Effect of Plasminogen on Body Weight of Diabetic Mice

Twenty male db/db mice aged 24-25 weeks were randomly divided into twogroups, ten in the control group administered with vehicle PBS and tenin the group administered with plasminogen, respectively. The day whenthe experiment began was recorded on Day 0, and the mice were weighedand grouped. From the second day of the experiment, plasminogen or PBSwas administered to the mice for 31 consecutive days, and the day wasrecorded as Day 1. Mice in the group administered with plasminogen wereinjected with plasminogen at a dose of 2 mg/0.2 mL/mouse/day via thetail vein, and an equal volume of PBS was administered to mice in thecontrol group administered with vehicle PBS. The mice were weighted ondays 0, 4, 7, 11, 16, 21, 26 and 31.

The results showed that there was no significant difference in bodyweight between mice in the group administered with plasminogen and thosein the control group administered with vehicle PBS on days 0, 4, 7, 11,16, 21, 26 and 31 (FIG. 4), indicating that plasminogen has littleeffect on animal body weight.

Example 5. Protective Effect of Plasminogen on Late Hepatic TissueInjury of Mice with Diabetic Hepatic Injury

Ten male db/db mice aged 24-25 weeks were randomly divided into twogroups, five in the control group administered with vehicle PBS and fivein the group administered with plasminogen, respectively. The day whenthe experiment began was recorded on Day 0, and the mice were weighedand grouped. From the second day of the experiment, plasminogen or PBSwas administered to the mice for 31 consecutive days, and the day wasrecorded as Day 1. Mice in the group administered with plasminogen wereinjected with plasminogen at a dose of 2 mg/0.2 mL/mouse/day via thetail vein, and an equal volume of PBS was administered to mice in thecontrol group administered with vehicle PBS. Mice were sacrificed on day32, and liver tissues were fixed in 10% neutral formalin fix solutionfor 24-48 hours. The fixed liver tissues were paraffin-embedded afterdehydration with alcohol gradient and permeabilization with xylene. Thethickness of the tissue sections was 5 μm. The sections were dewaxed andrehydrated, stained with hematoxylin and eosin (HE staining),differentiated with 1% hydrochloric acid in alcohol, and returned toblue with ammonia water. The sections were sealed after dehydration withalcohol gradient.

The HE staining results showed that in mice in the control groupadministered with vehicle PBS (FIG. 5A), the liver showed severesteatosis and lipid deposition, and fusion into large fat vacuoles, cellnuclei were squeezed to the edge (□), the hepatic cord was disordered,the hepatic sinus was narrowed, and there were different numbers ofinflammatory foci at the hepatic cord (↑);

and in mice in the group administered with plasminogen (FIG. 5B), theliver showed mild steatosis, mainly mild hydropic degeneration at theinjury, and dissolved cytoplasm (□), which were mainly distributed inthe area between the portal area and the central veins; the area aroundthe portal area and the central veins were affected mildly; and at thesame time, mild inflammatory cell infiltration was seen at the hepaticcord. This indicated that hepatic injury is obviously repaired afteradministration of plasminogen.

Example 6. Plasminogen Reduces the Fibrin Level in Liver Tissues ofDiabetic Mice

Ten male db/db mice aged 24-25 weeks were randomly divided into twogroups, five in the control group administered with vehicle PBS and fivein the group administered with plasminogen, respectively. The day whenthe experiment began was recorded on Day 0, and the mice were weighedand grouped. From the second day of the experiment, plasminogen or PBSwas administered to the mice for 31 consecutive days, and the day wasrecorded as Day 1. Mice in the group administered with plasminogen wereinjected with plasminogen at a dose of 2 mg/0.2 mL/mouse/day via thetail vein, and an equal volume of PBS was administered to mice in thecontrol group administered with vehicle PBS. Mice were sacrificed on day32, and liver tissues were fixed in 10% neutral formalin fix solutionfor 24 hours. The fixed liver tissues were paraffin-embedded afterdehydration with alcohol gradient and permeabilization with xylene. Thethickness of the tissue sections was 5 μm. The sections were dewaxed andrehydrated and washed with water once. The sections were incubated with3% hydrogen peroxide for 15 minutes and wash with water twice for 5minutes each time. The sections were blocked with 10% normal goat serumsolution (Vector laboratories, Inc., USA) for 1 hour; and after the timewas up, the goat serum solution was discarded, and the tissues werecircled with a PAP pen. The sections were incubated with rabbitanti-mouse fibrin (fibrinogen) antibody (Abcam) overnight at 4° C. andwashed with TBS twice for 5 minutes each time. The sections wereincubated with a secondary antibody, goat anti-rabbit IgG (HRP) antibody(Abcam), for 1 hour at room temperature and washed with TBS twice for 5minutes each time. The sections were developed with a DAB kit (Vectorlaboratories, Inc., USA). After washing with water three times, thesections were counterstained with hematoxylin for 30 seconds and flushedwith running water for 5 minutes. After gradient dehydration,permeabilization and sealing, the sections were observed under amicroscope at 200×.

Fibrinogen is the precursor of fibrin, and in the presence of tissueinjury, as a stress response to the body's injury, fibrinogen ishydrolyzed into fibrin^([12-14]). Therefore, the fibrinogen level can beused as a sign of the degree of injury.

The study found that compared with mice in the control groupadministered with vehicle PBS (FIG. 6A), those in the group administeredwith plasminogen (FIG. 6B) had a remarkably lower fibrin level in theliver tissue, indicating that injection of plasminogen can significantlyreduce the deposition of fibrin in diabetic mice, reflecting thesignificant repair function of plasminogen on the body's injury ofdiabetic mice.

Example 7. Plasminogen Reduces Inflammation of Liver Tissues of DiabeticMice

Ten male db/db mice aged 24-25 weeks were randomly divided into twogroups, five in the control group administered with vehicle PBS and fivein the group administered with plasminogen, respectively. The day whenthe experiment began was recorded on Day 0, and the mice were weighedand grouped. From the second day of the experiment, plasminogen or PBSwas administered to the mice for 31 consecutive days, and the day wasrecorded as Day 1. Mice in the group administered with plasminogen wereinjected with plasminogen at a dose of 2 mg/0.2 mL/mouse/day via thetail vein, and an equal volume of PBS was administered to mice in thecontrol group administered with vehicle PBS. Mice were sacrificed 31days after administration of plasminogen, and liver tissues were fixedin 10% neutral formalin fix solution for 24 hours. The fixed livertissues were paraffin-embedded after dehydration with alcohol gradientand permeabilization with xylene. The thickness of the tissue sectionswas 5 μm. The sections were dewaxed and rehydrated and washed with wateronce. The sections were incubated with 3% hydrogen peroxide for 15minutes and wash with water twice for 5 minutes each time. The sectionswere blocked with 10% normal goat serum for 1 hour, and after the timewas up, the serum was thrown away, and the tissues were circled with aPAP pen. The sections were incubated with a rabbit polyclonal antibodyagainst F4/80 (Abcam) overnight at 4° C. and washed with TBS twice for 5minutes each time. The sections were incubated with a secondaryantibody, goat anti-rabbit IgG (HRP) antibody (Abcam), for 1 hour atroom temperature and washed with TBS twice. The sections were developedwith a DAB kit (Vector laboratories, Inc., USA). After washing withwater three times, the sections were counterstained with hematoxylin for30 seconds and flushed with running water for 5 minutes. After gradientdehydration, permeabilization and sealing, the sections were observedunder a microscope at 400×.

F4/80 is a macrophage marker that can indicate the extent and stage ofan inflammatory response. The results showed that compared with mice inthe control group administered with vehicle PBS (FIG. 7A), the F4/80positive level was significantly reduced in those in the groupadministered with plasminogen (FIG. 7B), indicating that inflammation ofthe liver tissues is reduced after administration of plasminogen. FIG.7C shows the results of quantitative analysis of F4/80immunohistochemical positive expression, in which the expression ofF4/80 in mice in the group administered with plasminogen wassignificantly reduced with statistical difference, indicating thatinjection of plasminogen can significantly promote the repair of liverinflammation of diabetic mice.

Example 8. Plasminogen Promotes the Repair of Liver Injury of DiabeticMice

Nine male db/db mice aged 25-28 weeks were randomly divided into twogroups, three in the control group administered with vehicle PBS and sixin the group administered with plasminogen, respectively. The day whenthe experiment began was recorded on Day 0, and the mice were weighedand grouped. From the second day of the experiment, plasminogen or PBSwas administered to the mice for 31 consecutive days, and the day wasrecorded as Day 1. Mice in the group administered with plasminogen wereinjected with plasminogen at a dose of 2 mg/0.2 mL/mouse/day via thetail vein, and an equal volume of PBS was administered to mice in thecontrol group administered with vehicle PBS. Whole blood was taken fromthe removed eyeballs 31 days after administration of plasminogen. Afterthe serum was precipitated, it was centrifuged at 3500 r/min for 10minutes at 4° C., and the supernatant was taken for detection. In thisexperiment, the content of alanine transaminase (ALT) in serum wasdetected by Reitman-Frankel colorimetry using an alanine transaminasedetection kit (Nanjing Jiancheng Biological Engineering ResearchInstitute, Catalog No. C009-2).

Alanine transaminase is an important index of liver healthstatus^([15,16]), and the normal reference value interval of alaninetransaminase is 9-50 U/L. The detection results showed that the ALTcontent in serum of mice in the control group administered with vehiclePBS was significantly higher than the normal physiological index,whereas the content in mice in the group administered with plasminogenhad returned to normal levels in the body; and the content in mice inthe group administered with plasminogen was significantly lower thanthat in mice in the control group administered with vehicle PBS, andthere was a statistical difference (FIG. 8). This indicated thatinjection of plasminogen can effectively repair the liver injury inmodel mice with late diabetic diabetes.

Example 9. Protective Effect of Plasminogen on the Liver in Case ofAcute Hepatic Poisoning

Eighteen male or female 7-8-week-old plg^(−/−) mice were randomlydivided into two groups, nine in the control group administered withvehicle PBS and nine in the group administered with plasminogen,respectively. Two groups of mice were administered with carbontetrachloride via intraperitoneal injection at 0.5 mL/kg body weight for2 consecutive days to establish an acute hepatic injury model^([17,18]).Carbon tetrachloride should be diluted with corn oil before use, and thevolume ratio of the former to the latter is 1:7. The day of modelestablishment was day 0, and plasminogen or PBS was administered to themice from day 1. Mice in the group administered with plasminogen wereadministered with plasminogen at a dosage of 1 mg/0.1 mL/mouse/day, andan equal volume of PBS was administered to mice in the control groupadministered with vehicle PBS, both for 7 consecutive days. Three micefrom both groups were respectively sacrificed on days 0, 2 and 7, themice were dissected and the liver conditions were observed and recorded,and then liver tissues were fixed in 10% neutral formalin fix solutionfor 24-48 hours. The fixed liver tissues were paraffin-embedded afterdehydration with alcohol gradient and permeabilization with xylene. Thethickness of the tissue sections was 5 μm. The sections were dewaxed andrehydrated, stained with hematoxylin and eosin (HE staining),differentiated with 1% hydrochloric acid in alcohol, and returned toblue with ammonia water. The sections were sealed after dehydration withalcohol gradient and observed under a microscope at 200×.

The HE staining results showed that on day 0, the livers of mice in thecontrol group administered with vehicle PBS (FIGS. 9A-C) and the groupadministered with plasminogen (FIGS. 9D-F) mainly showed fragmentednecrosis around the central veins, in which cell nuclei were fragmentedand cytoplasm was stained lightly in the necrotic area, and there weremoderate hydropic degeneration and cellular edema also occurring inother non-necrotic areas. On day 2, the central veins expanded, thestructures of hepatocytes were disordered, and there was a littleinflammatory cell infiltration. There was no significant differencebetween two groups. However, on day 7, mice in the control groupadministered with vehicle PBS still had a little hepatocytedegeneration, mild cellular edema, disordered hepatic cord and narrowedhepatic sinus, and there was mild inflammatory cell infiltration aroundthe portal area; whereas the liver of mice in the group administeredwith plasminogen had basically returned to normal cytoplasm redstaining, and had regular hepatic cord and clear hepatic sinus. Thisindicated that plasminogen can promote the repair of hepatic injury.

Example 10. Protective Effect of Plasminogen on the Liver in Case ofAcute Hepatic Poisoning

Eighteen male 7-11-week-old plg^(−/−) mice were randomly divided intotwo groups, nine in the control group administered with vehicle PBS andnine in the group administered with plasminogen, respectively. Twogroups of mice were administered with carbon tetrachloride viaintraperitoneal injection at 0.5 mL/kg body weight once to establish anacute hepatic injury model^([17,18]). Carbon tetrachloride should bediluted with corn oil before use, and the volume ratio of the former tothe latter is 1:7. Plasminogen or vehicle PBS was administered to themice within half an hour after completion of model establishment. Micein the group administered with plasminogen were injected withplasminogen at a dosage of 1 mg/0.1 mL/mouse/day, and an equal volume ofPBS was administered to mice in the control group administered withvehicle PBS, both for 2 consecutive days. Three mice from both groupswere respectively sacrificed at hours 18, 24 and 48 afteradministration, the mice were dissected and the liver conditions wereobserved and recorded, and then liver tissues were fixed in 10% neutralformalin fix solution for 24-48 hours. The fixed liver tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The thickness of the tissue sections was 5μm. The sections were dewaxed and rehydrated, stained with hematoxylinand eosin (HE staining), differentiated with 1% hydrochloric acid inalcohol, and returned to blue with ammonia water. The sections weresealed after dehydration with alcohol gradient and observed under amicroscope at 200×.

The results showed that mice in the control group administered withvehicle PBS (FIGS. 10A-C) showed different levels of necrosis at 18 h,24 h and 48 h, and mainly fragmented necrosis at 18 h and 24 h, andbridging necrosis, nuclear fragmentation and lightly stained cytoplasmoccurring at 48 h; the injury was progressively worsening and mainlyaround the central veins; and there was moderate inflammatory cellinfiltration in the necrotic area (↓), and there was mild necrosis,mainly mild hydropic degeneration around the portal area, accompanied bymild inflammatory cell infiltration and mild bile duct hyperplasia (□);and compared with mice in the control group, the mice in the groupadministered with plasminogen (FIGS. 10D-F) showed no apparent necrosisat 18 h, 24 h and 48 h; the injury was mainly mild hydropic degenerationand distributed around the portal area; hepatocytes around the centralveins were not affected; and these conditions were better at 24 h thanthose at 18 h, in which hydropic degeneration was reduced, hepatocytesaround the central veins showed mild steatosis and lightly stainedcytoplasm, both accompanied by mild inflammatory cell infiltration. Thisindicated that plasminogen can promote the repair of hepatic injury ofplg′ model mice with acute hepatic injury.

Example 11. Plasminogen Reduces Fibrin Deposition in the Liver Tissuesof Model Mice with Acute Hepatic Injury

Eighteen male 7-11-week-old plg^(−/−) mice were randomly divided intotwo groups, nine in the control group administered with vehicle PBS andnine in the group administered with plasminogen, respectively. Twogroups of mice were administered with carbon tetrachloride viaintraperitoneal injection at 0.5 mL/kg body weight once to establish anacute hepatic injury model^([17,18]). Carbon tetrachloride should bediluted with corn oil before use, and the volume ratio of the former tothe latter is 1:7. Plasminogen or vehicle PBS was administered to themice within half an hour after completion of model establishment. Micein the group administered with plasminogen were injected withplasminogen at a dosage of 1 mg/0.1 mL/mouse/day, and an equal volume ofPBS was administered to mice in the control group administered withvehicle PBS, both for 2 consecutive days. Three mice from both groupswere respectively sacrificed at hours 18, 24 and 48 afteradministration, the mice were dissected and the liver conditions wereobserved and recorded, and then liver tissues were fixed in 10% neutralformalin fix solution for 24-48 hours. The fixed liver tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The thickness of the tissue sections was 5μm. The sections were dewaxed and rehydrated and washed with water once.The sections were incubated with 3% hydrogen peroxide for 15 minutes andwash with water twice for 5 minutes each time. The sections were blockedwith 10% normal goat serum solution (Vector laboratories, Inc., USA) for1 hour; and after the time was up, the goat serum solution wasdiscarded, and the tissues were circled with a PAP pen. The sectionswere incubated with rabbit anti-mouse fibrin (fibrinogen) antibody(Abcam) overnight at 4° C. and washed with TBS twice for 5 minutes eachtime. The sections were incubated with a secondary antibody, goatanti-rabbit IgG (HRP) antibody (Abcam), for 1 hour at room temperatureand washed with TBS twice for 5 minutes each time. The sections weredeveloped with a DAB kit (Vector laboratories, Inc., USA). After washingwith water three times, the sections were counterstained withhematoxylin for 30 seconds and flushed with running water for 5 minutes.After gradient dehydration, permeabilization and sealing, the sectionswere observed under a microscope at 200×.

Fibrinogen is the precursor of fibrin, and in the presence of tissueinjury, as a stress response to the body's injury, fibrinogen ishydrolyzed into fibrin^([12-14]). Therefore, the fibrinogen level can beused as a sign of the degree of injury.

The results showed that at three time points, 18 h, 24 h and 48 h, micein the group administered with plasminogen (FIGS. 11D-F) showedsignificantly lighter fibrin-positive staining than that in mice in thecontrol group administered with vehicle PBS (FIGS. 11A-C), and thefibrin staining also tended to become lighter gradually with theextension of time. This indicated that injection of plasminogen canreduce fibrin deposition and promote the repair of hepatic injury.

Example 12. Plasminogen Promotes the Repair of Liver Inflammation ofMice Irradiated with 5.0 Gy X-Rays

In this experiment, ten healthy 6-8-week-old male C57 mice were used andrandomly divided into two groups, five in the control group administeredwith vehicle PBS and five in the group administered with plasminogen,respectively. After the grouping was completed, a radiation-inducedinjury model was established by uniformly irradiating the mice with 6 MVX-rays from a linear accelerator at 5.0 Gy once systemically, in whichthe absorbed dosage rate was 2.0 Gy/min and the absorbed dosage was 5.0Gy (irradiation for 2.5 minutes). Plasminogen was administered to themice within 3 hours after the model was established. The day when theexperiment began was Day 0, and the mice were weighed and grouped. Themice were treated with radiation and administered with plasminogen orvehicle PBS from day 1. The administration period was 10 days. After thecompletion of the administration, the medication of animals wasdiscontinued and they were observed for 11 days. The entire experimentalperiod was 21 days. Mice in the group administered with plasminogen wereinjected with plasminogen at a dosage of 1 mg/0.1 mL/mouse/day via thetail vein, and an equal volume of PBS was administered to mice in thecontrol group administered with vehicle PBS. Mice were sacrificed anddissected on day 21, and livers were fixed in 10% neutral formalin fixsolution for 24-48 hours. The fixed liver tissues were paraffin-embeddedafter dehydration with alcohol gradient and permeabilization withxylene. The thickness of the tissue sections was 5 μm. The sections weredewaxed and rehydrated and washed with water once. The sections wererepaired with Tris-EDTA for 30 minutes, and gently rinsed with waterafter cooling at room temperature for 20 minutes. The sections wereincubated with 3% hydrogen peroxide for 15 minutes, and the tissues werecircled with a PAP pen. The sections were blocked with 10% normal goatserum (Vector laboratories, Inc., USA) for 1 hour; and after the timewas up, the goat serum solution was discarded. The sections wereincubated with rabbit anti-mouse F4/80 antibody (Abcam) at 4° C.overnight and washed with TBS twice for 5 minutes each time. Thesections were incubated with a secondary antibody, goat anti-rabbit IgG(HRP) antibody (Abcam), for 1 hour at room temperature and washed withTBS twice for 5 minutes each time. The sections were developed with aDAB kit (Vector laboratories, Inc., USA). After washing with water threetimes, the sections were counterstained with hematoxylin for 30 secondsand flushed with running water for 5 minutes. After gradientdehydration, permeabilization and sealing, the sections were observedunder a microscope at 200×.

The F4/80 immunohistochemical results showed that after modelestablishment with 5.0 Gy X-ray irradiation, the expression level of themacrophage marker in mice in the control group administered with vehiclePBS (FIG. 12A) was higher than that of the macrophage marker in mice inthe group administered with plasminogen (FIG. 12B), indicating that theinflammation of the liver tissues of the animals was significantlyreduced after administration of plasminogen.

Example 13. Plasminogen Reduces Fibrin Deposition in the Liver Tissuesof Model Mice with Injury Induced by Cisplatin Chemotherapy

Ten healthy 8-9-week-old male C57 mice were used and randomly dividedinto two groups, five in the control group administered with vehicle PBSand five in the group administered with plasminogen, respectively. Afterthe grouping was completed, a chemotherapy-induced injury model wasestablished by single intraperitoneal injection of cisplatin at 10 mg/Kgbody weight. After the model was established, mice in the groupadministered with plasminogen were administered with plasminogen at adosage of 1 mg/mouse/day via tail vein injection, and an equal volume ofPBS was administered to mice in the control group administered withvehicle PBS. The day when the experiment began was Day 0, and the micewere weighed and grouped. The mice were injected with cisplatinintraperitoneally from day 1 for model establishment. Plasminogen orvehicle PBS was administered to the mice within 3 hours after completionof model establishment, and the administration period was 7 days. Micewere sacrificed on day 8, and livers were fixed in 10% neutral formalinfix solution for 24-48 hours. The fixed liver tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The thickness of the tissue sections was 5μm. The sections were dewaxed and rehydrated and washed with water once.The sections were repaired with citric acid for 30 minutes, and gentlyrinsed with water after cooling at room temperature for 10 minutes. Thesections were incubated with 3% hydrogen peroxide for 15 minutes, andthe tissues were circled with a PAP pen. The sections were blocked with10% normal goat serum (Vector laboratories, Inc., USA) for 1 hour; andafter the time was up, the goat serum solution was discarded. Thesections were incubated with rabbit anti-mouse fibrin antibody (Abcam)overnight at 4° C. and washed with TBS twice for 5 minutes each time.The sections were incubated with a secondary antibody, goat anti-rabbitIgG (HRP) antibody (Abcam), for 1 hour at room temperature and washedwith TBS twice for 5 minutes each time. The sections were developed witha DAB kit (Vector laboratories, Inc., USA). After washing with waterthree times, the sections were counterstained with hematoxylin for 30seconds and flushed with running water for 5 minutes. After gradientdehydration, permeabilization and sealing, the sections were observedunder a microscope at 200×.

Fibrinogen is the precursor of fibrin, and in the presence of tissueinjury, as a stress response to the body's injury, fibrinogen ishydrolyzed into fibrin^([12-14]). Therefore, the fibrinogen level can beused as a sign of the degree of injury.

The results showed that fibrin-positive staining in the liver tissues ofmice in the control group administered with vehicle PBS (FIG. 13A) wassignificantly darker than that in the liver tissues of mice in the groupadministered with plasminogen (FIG. 13B). This indicated thatplasminogen can significantly reduce fibrin deposited in injured livertissues, indicating that plasminogen can promote the repair of hepaticinjury caused by the chemotherapy drug cisplatin.

Example 14. Protective Effect of Plasminogen on the Liver in Case ofAcute Hepatic Poisoning

Six male 7-11-week-old plg^(−/−) mice were randomly divided into twogroups, three in the control group administered with vehicle PBS andthree in the group administered with plasminogen, respectively. Twogroups of mice were administered with carbon tetrachloride viaintraperitoneal injection at 0.5 mL/kg body weight once to establish anacute hepatic injury model^([17,18]). Carbon tetrachloride should bediluted with corn oil before use, and the volume ratio of the former tothe latter is 1:7. Plasminogen or vehicle PBS was administered to themice within half an hour after completion of model establishment. Micein the group administered with plasminogen were administered withplasminogen at a dosage of 1 mg/0.1 mL/mouse/day, and an equal volume ofPBS was administered to mice in the control group administered withvehicle PBS, both for 7 consecutive days. Mice were sacrificed anddissected on day 8, the liver conditions were observed and recorded, andthen liver tissues were fixed in 10% neutral formalin fix solution for24-48 hours. The fixed liver tissues were paraffin-embedded afterdehydration with alcohol gradient and permeabilization with xylene. Thethickness of the tissue sections was 5 μm. The sections were dewaxed andrehydrated, stained with hematoxylin and eosin (HE staining),differentiated with 1% hydrochloric acid in alcohol, and returned toblue with ammonia water. The sections were sealed after dehydration withalcohol gradient and observed under a microscope at 200×.

The results showed that in the livers of mice in the control groupadministered with vehicle PBS (FIG. 14A), the central veins expanded,the endothelial cells were necrotic, hepatocytes around the centralveins all had large areas of focal necrosis with fragmented and darklystained nuclei, and there were mild hydropic degeneration, cellularedema and clear cytoplasm in other non-necrotic areas, accompanied bymild inflammatory cell infiltration in the necrotic area; and in thelivers of mice in the group administered with plasminogen (FIG. 14B),there was no apparent necrosis, injury was mainly mild hydropicdegeneration, and there were enhanced acidophily and red staining in thecytoplasm of a small amount of hepatocytes. Injury of mice in the groupadministered with plasminogen was obviously milder than that of mice inthe control group administered with vehicle PBS, indicating thatplasminogen can promote the repair of hepatic injury of plg^(−/−) modelmice with acute hepatic injury.

REFERENCES

-   [1] Xiao-Lan Lu, Jin-Yan Luo, Ming Tao, Yan Gen, Ping ZHAO, Hong-Li    Zhao, Xiao-Dong Zhang, Nei Dong, Risk factors for alcoholic liver    disease in China. World Journal of Gastroenterology. 2004, 10(16)-   [2] Tim C M A Schreuder, Bart J Verwer, Carin M J van Nieuwkerk,    Chris J J Mulder, Nonalcoholic fatty liver disease: An overview of    current insights in pathogenesis, diagnosis and treatment. World    Journal of Gastroenterology. 2011, 14(16)-   [3] Wiman, B. and Wallen, P. (1975). Structural relationship between    “glutamic acid” and “lysine” forms of human plasminogen and their    interaction with the NH2-terminal activation peptide as studied by    affinity chromatography. Eur. J. Biochem. 50, 489-494.-   [4] Saksela, O. and Rifkin, D. B. (1988). Cell-associated    plasminogen activation: regulation and physiological functions.    Annu. Rev. Cell Biol. 4, 93-126-   [5] Raum, D., Marcus, D., Alper, C. A., Levey, R., Taylor, P. D.,    and Starzl, T. E. (1980). Synthesis of human plasminogen by the    liver. Science 208, 1036-1037-   [6] Wallén P (1980). Biochemistry of plasminogen. In Fibrinolysis,    Kline D L and Reddy K K N, eds. (Florida: CRC.-   [7] Sottrup-Jensen, L., Zajdel, M., Claeys, H., Petersen, T. E., and    Magnusson, S. (1975). Amino-acid sequence of activation cleavage    site in plasminogen: homology with “pro” part of prothrombin. Proc.    Natl. Acad. Sci. U. S. A 72, 2577-2581.-   [8] Marder V J, Novokhatny V. Direct fibrinolytic agents:    biochemical attributes, preclinical foundation and clinical    potential [J]. Journal of Thrombosis and Haemostasis, 2010, 8(3):    433-444.-   [9] Hunt J A, Petteway Jr S R, Scuderi P, et al. Simplified    recombinant plasmin: production and fu-nctional comparison of a    novel thrombolytic molecule with plasma-derived plasmin [J]. Thromb    Haemost, 2008, 100(3): 413-419.-   [10] Sottrup-Jensen L, Claeys H, Zajdel M, et al. The primary    structure of human plasminogen: Isolation of two lysine-binding    fragments and one “mini”-plasminogen (MW, 38, 000) by    elastase-catalyzed-specific limited proteolysis [J]. Progress in    chemical fibrinolysis and thrombolysis, 1978, 3: 191-209.-   [11] Nagai N, Demarsin E, Van Hoef B, et al. Recombinant human    microplasmin: production and potential therapeutic properties[J].    Journal of Thrombosis and Haemostasis, 2003, 1(2): 307-313.-   [12] Jae Kyu Ryu, Mark A. Petersen, Sara G. Murray et al. Blood    coagulation protein fibrinogen promotes autoimmunity and    demyelination via chemokine release and antigen presentation. NATURE    COMMUNICATIONS, 2015, 6:8164.-   [13] Dimitrios Davalos Katerina Akassoglou. Fibrinogen as a key    regulator of inflammation in disease. Seminars in    Immunopathology, 2012. 34(1):43-62.-   [14] Valvi D, Mannino D M, Mullerova H, et al. Fibrinogen, chronic    obstructive pulmonary disease (COPD) and outcomes in two United    States cohorts. Int J Chron Obstruct Pulmon Dis 2012; 7:173-82.-   [15] Karmen A, Wroblewski F, Ladue J S (January 1955). Transaminase    activity in human blood. The Journal of Clinical Investigation. 34    (1): 126-31.-   [16] Wang C S, Chang T T, Yao W J, Wang S T, Chou. P (April 2012).    Impact of increasing alanine aminotransferase levels within normal    range on incident diabetes. Journal of the Formosan Medical    Association=Taiwan Yi Zhi. 111 (4): 201-8.-   [17] Hua Liu, Zhe Wang, Michael J Nowicki. Caspase-12 mediates    carbon tetrachloride-induced hepatocyte apoptosis in mice. World J    Gastroenterol 2014 Dec. 28; 20(48): 18189-18198.-   [18] Kamyar Zahedi, Sharon L. Barone et al. Hepatocyte-specific    ablation of spermine/spermidine-N1-acetyltransferase gene reduces    the severity of CCl4-induced acute liver injury. Am J Physiol    Gastrointest Liver Physiol 303: G546-G560, 2012.

1. A method for preventing and/or treating hepatic tissue injury and itsrelated disorders in a subject, comprising administering an effectiveamount of plasminogen to the subject.
 2. The method of claim 1, whereinthe hepatic tissue injury and its related disorders are hepatic injuryand its related disorders caused by radiation or chemical substances. 3.The method of claim 1, wherein the hepatic tissue injury and its relateddisorders are toxic hepatic injury and its related disorders.
 4. Themethod of claim 1, wherein the hepatic tissue injury and its relateddisorders are diabetic hepatic injury and its related disorders.
 5. Themethod according to claim 4, wherein the diabetic hepatic injury and itsrelated disorders are caused by diabetes mellitus-induced angiopathy oflarge vessels, small vessels and microvessels.
 6. The method of claim 1,wherein the hepatic tissue injury and its related disorders comprisehepatic dysfunction, abnormal hepatic enzymology, liver discomfort andhaphalgesia, hepatomegaly, splenomegaly, hepatosplenomegaly, hepatitis,fatty liver, cholangitis, hepatic cirrhosis, hepatic necrosis andhepatic carcinoma caused by hepatic tissue injury.
 7. The method ofclaim 1, wherein the plasminogen has at least 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% sequence identity with SEQ ID No. 2, 6, 8, 10 or 12, andstill has the plasminogen activity.
 8. The method of claim 1, whereinthe plasminogen is a protein that comprises a plasminogen activefragment and still has the plasminogen activity.
 9. The method of claim1, wherein the plasminogen is selected from Glu-plasminogen,Lys-plasminogen, mini-plasminogen, micro-plasminogen, δ(delta)-plasminogen or any combination thereof.
 10. The method of claim1, wherein the plasminogen is administered in combination with one ormore other drugs.
 11. An article for preventing and/or treating hepatictissue injury and its related disorders in a subject, comprising acontainer containing an effective dosage of plasminogen, andinstructions for directing the administration of the article to preventand/or treat hepatic tissue injury and its related disorders in thesubject.
 12. The article of claim 11, further comprising a containercontaining one or more other drugs.
 13. The article of claim 12, whereinthe instructions further indicate that the plasminogen is administeredbefore, simultaneously with and/or after the administration of the otherdrugs.